Microdelivery device and method for enhanced drug administration to the eye

ABSTRACT

A device for pinpoint application of micro-quantities of a pharmacologically suitable composition to the outer hard coat of the eye, comprises a micro-container with a fixation element. The micro-container comprises a bore whose aperture is adapted to contact the eye surface, and the fixation element is adapted for reversibly adhering to the eye surface.

The present invention concerns a device and method of application forpinpoint delivery of micro-quantities of pharmaceutical compositionssuch as collagenase to a predetermined location on the outer hard coatof the eye, resulting in increased permeability at that location. Themethod is useful in the treatment of glaucoma when applied to thelimbus, by increasing outflow facility. When treating other oculardisorders, the method may be applied at other locations of the sclera toenhance penetration into the eye of periocularly administered drugs.

BACKGROUND OF THE INVENTION

Collagen is the major constituent of the tissues forming the eye coat(Collagenase, Mandel, I. "Collagenase comes of age", p.1, Gordon andBreech, Science Publishers, Inc. 1972). Enzymes such as collagenase,which degrade collagen, are of importance in controlling conditionsinvolving the collagen-rich tissue.

The use of enzymes in the medical field is well known. For example,alpha-chymotrypsin was used to lyse zonules in cataract surgery(I.C.C.E), hyaluronidase is used extraocularly as a means for spreadinglocal anesthesia more effectively through tissue, and Tissue PlasminogenActivator has been proposed for clot-removal and filtering blebreformation. The use of proteolytic enzymes as biopolymeric drycomposition for treatment of wounds has been reported in U.S. Pat. No.4,613,502. Detoxified agents such as enzymes obtained from snake venomhave been used in the treatment of ocular disorders as disclosed in U.S.Pat. No. 3,869,548. Another bacterial extract, the toxin fromClostridium botulinus was proposed by Scott (J. Ped. Opthal. 17;21) in1980 for use in ophthamology replacing strabismus surgery.

The use of collagenase has been disclosed in the art. Processes forproduction of collagenase from the bacterium Clostridium histolyticumhave been disclosed in U.S. Pat. Nos. 3,705,083 and 3,821,364. Processesfor preparing other collagenase preparations have been disclosed in U.S.Pat. Nos. 3,267,006 and 3,677,900. Collagenase has been used in thetreatment of herniated intervertebral disc and such treatment isdisclosed in U.S. Pat. No. 3,678,158. Use of collagenase as an adjunctto vitrectomy with membranectomy was proposed in U.S. Pat. No.3,678,158. Use of collagenase has been described as an inhibitor ofcollagen-induced platelet aggregation and to be useful in selectivedegradation of collagen in the eye to remove scar tissue (EuropeanPatent Application No. 233,908). Collagenase obtained from Vibrioalginolyticus is said to be useful to prevent formation of deep scarsduring healing of burns and other lesions and has been proposed to treatdental caries, dental pulp and skin burns (U.S. Pat. No. 4,732,758).Soviet Union Patent No. 1,286,195 describes a method of treatment ofglaucoma involving the formation of paths of outflow of intraocularfluid by direct injection of a proteolytic enzyme, leucozyme, into thesclera 3 mm posteriorly to the limbus.

The following brief discussion concerning basic ocular histology,anatomy and ophthalmic surgery serves to describe the collagen richcomposition of the sclera and limbus, their involvement in oculardiseases such as glaucoma and the problems facing ophthalmic surgeonswho are concerned with the treatment of it. Basically the hard outerlayer of the mammal/an eyeball is formed by the opaque white sclera(83%) continued anteriorly by the cornea (16%) and posteriorly by theoptic nerve sheath (<1%). These are densely collagenous hypocellularstructures composed of (75%) of hydroxyproline-rich collagen, elastictissue and mucopolysaccharides. The collagen fiber diameter variesbetween 28-300 nm with a periodicity of 64 nm. Embedded inmucopolysaccharide rich substance, the bundles are approximatelyparallel to the surface which in the cornea have a strict layering,responsible for its vital transparency. In the sclera and sclerocornealjunction the inner collagen bundles near, the Schlem canal arerelatively inactive metabolically, having no intrinsic capillary bed andonly a few fibrocytes. Between the transparent cornea anteriorly and theiris posteriorly is the anterior chamber which contains aqueous humor.The anterior chamber is directly connected with the small posteriorchamber of the eye via the pupillary opening. Aqueous humor is secretedby cells of the ciliary body. This fluid flows into the posteriorchamber and through the pupil into the anterior chamber, in order tonourish the cornea and maintain the internal ocular pressure. In humans,this aqueous fluid is formed at an approximate rate of 2.1microliters/minute. The volume of the anterior chamber is 0.25 ml andthis fluid which flows into the chamber is filtered out partiallythrough the uveoscleral path (10-20%) but mainly through a system ofchannels in the trabecular meshwork (80-90%). The trabecular meshwork isa system of filters located in the angle of the anterior chamber underthe sclerocorneal junction, the limbus. This meshwork consists of acollection of collagenous pillars which are lined by endothelial cells.It is the trabecular meshwork through which the aqueous fluid flows byentering a gradually enlarging system of collector channels to enter theaqueous veins and ultimately leave the anterior portion of the eye.Obstruction of this outflow system is thought to be the main reason forthe increased intraocular pressure observed in glaucoma.

Movement of aqueous solution containing ions and solutes, smaller thanor about equal in size to serum albumin, through the sclera results froma pressure difference between the suprachoroidal space where thepressure is about 1-2 mm Hg below the intraocular pressure and theepiscleral venous pressure which approaches 10 mm Hg. This transscleralmovement is slowed due to an eventual decrease in intraocular pressureand increases in pathologic cases such as glaucoma where the pressure ishigh.

Simple glaucoma is a genetically determined, age-related, oculardisease. It is one of the world's leading causes of blindness and in theU.S.A encompasses about 67,000 legally blind with an incidence of 5,500new cases each year and 12% of all new cases of blindness. Glaucoma anddirectly related conditions account for 2 million suffers, 900,000visually impaired, and 8.5% of all visits to an ophthalmologist, with a440 million dollars direct and 1.9 billion dollars indirect health cost.(National Society for Prevention of Blindness, Vision Problems in U.S.A.Data Analysis 1980). It is rare in children but becomes increasinglyprevalent with each decade over the age of 40 (0.4%-1.6%). Many more areat risk of developing glaucomatous loss of vision.

Glaucoma develops in two separate but related pathogenic stages. First,the ocular drainage of aqueous humor becomes impaired and the outflowfacility decreases from about 0.3 to 0.05 microliter/minute/mmHg,establishing an inflow/outflow balance at a higher intraocular pressure.Second, the high intraocular pressure, aided by other less well definedpathogenic factors, causes compressive damage to fibers of the opticnerve head, and thereby insidious progression toward blindness due toloss of visual field.

Glaucoma is treated nonsurgically and surgically, both with the aim ofachieving an increase in aqueous outflow or a decrease in its inflowwith consequent lowering of intraocular pressure. In mild and moderatecases treatment is based upon long term topical application of drops ofanti-glaucoma drugs, which however have a significant noxious effect asfirst reported by Barkan (Am. J. Opthamol.; 37:724) in 1954 and provenby histology of the tendon and the conjunctiva more than 30 years later.These effects obscure the prognosis of glaucoma surgery if eventuallyneeded in the future as reported by Sherwood (Invest. Ophthal. Vis. Sci.28 (suppl,135) in 1987. This encouraged clinical investigators torecommend surgery as the first stage of the glaucoma treatment (Migdaland Hitchings, Eye; 105653-656, 1986 and Lavin (Arch Ophthal; 108:1543in 1990). When drug treatment fails, invasive methods such as filtrationsurgery, cryoablations and laser trabeculoplasty, are employed. Newertreatments still at a clinical investigative stage includeintraoperative seton valve implantation, various types of laser surgery,thermal sclerostomy, ultrasonic disruption, and uncontrolled ocularinjection of enzymes employed. All are accompanied by several drawbackssuch as limited rate of success and even more, duraction of the effect(if any, in the case of ocular injection, according to Soviet UnionPatent No. 1,286,195, since it was not reproducible under similarconditions in an animal model).

The following brief discussion concerning the most common complicationsof the supramentioned methods of glaucoma treatment is necessary toemphasize the need for the new method of treatment provided by thepresent invention, as detailed hereafter. The first choice whenconsidering surgery is the angle laser treatment (trabeculoplasty). Withthis method it is possible to lower the intraocular pressure byincreasing the outflow from the anterior chamber. In this treatment,small cicatrizing areas of about 60 microns, created with a chromaticlaser, presumably enlarge the obstructed sclerotic trabecular pores.This treatment has various rates of success in different types ofglaucoma and is especially low in juvenile and neovascular types.Furthermore, its effect is limited to two years and can be repeated onlyonce. Other non mutilating surgical treatments are: Nd-Y.A.G.cyclophototherapy and cyclocryotherapy which may cause cataract andphthisis bulby and are therefore used only as a last resort at end-stageglaucoma, such as complicated, juvenile and neovascular types. Othermethods such as Nd-Y.A.G. laser angle surgery, ultrasonic disruption,thermal sclerostomy, dye contact laser sclerostomy and internalsclerostomy with automated trephine are all still under investigation.Other techniques include the invasive and mutilating filtrationsurgeries that frequently fail on follow up (2 to 5 years) ornecessitate in complicated cases, the use of large and partially toxicamounts of anti-metabolites, i.e. 5-fluorouracil and mitomycin or use ofsubconjunctivally implanted various seton valves. These procedures arerepeatable only once (in special circumstances twice) and are associatedwith a wide range of complications such as sudden permanent loss ofcentral vision, infection, malignant glaucoma, serious bleeding, flatanterior chamber (10%), failure of filtration (10%), technical problems,progression of glaucoma (15%) and progression of cataract (100%). Theperiocular injection of leucozyme (Soviet Union Patent Application No.1286195) is particularly hazardous because a major part of the ocularregion contains collagenous material. The uncontrolled injection ofleucozyme and its unmonitored contact with any portion of the ocularregion can risk continued vision in mammals and particularly in humans.Such injection cannot be directed to the limbus, since the nearby corneawould be severely damaged by lateral spreading of collagenase. Injectionto the sclera in a more posterior location was found ineffective inlowering the intraocular pressure in rabbits using collagenase insteadof the undefined leucozyme preparation.

SUMMARY OF THE INVENTION

The instant invention provides a solution to the many difficultiesfacing ophthalmologists engaging in the treatment of glaucoma byproviding a device and method for augmenting controlled local limbalpermeability with consequent increase of outflow facility accompanied bylowering of intraocular pressure, which is the aim of every glaucomatreatment. Furthermore, this invention provides a device and method fortreatment of other ocular disorders by enhancing the penetration oflarge amounts of therapeutic agents continuously and in a controlledmanner into the ocular cavity by means of direct contact with thesclera.

It is therefore the object of the present invention to provide a deviceand method for permeating small but effective amounts ofpharmacologically suitable compositions into the eye non-invasively.

More specifically, it is the object of the invention to provide a deviceand method for pinpoint delivery of pharmacologically suitablecompositions to the outer hard coat of the eye.

Another object of the invention is to provide a device and method fortreating glaucoma.

A further object of the present invention to provide a device and methodfor pinpoint application of collagenase to the limbus.

Yet another object of the invention is to provide a method and devicefor safe application of the device to different locations on the limbuswithout adverse effects.

A still further object is to provide a method and device for repeateddelivery of pharmaceutical compositions to the same location with thesame device.

In accordance with this invention therefore, there is provided a devicefor pinpoint application of micro-quantities of a pharmacologicallysuitable composition to a preselected portion of the outer hard coat ofthe eye, said device comprising a micro-container with fixation meansfor reversible attachment of said micro-container to the outer hard coatof the eye adjacent to the preselected portion thereof, saidmicro-container comprising a pharmaceutical composition delivery borewhose aperture is adapted to contact the preselected portion of theouter hard coat of the eye so as to expose only the preselected portionthereof to the pharmaceutical composition.

A method for administering pharmaceutical compositions to the eye withthe above device is also provided.

DETAILED DESCRIPTION OF THE DRAWINGS

The invention will best be understood with reference to the drawings inwhich

FIGS. 1a and 1b illustrate a cross-sectional and ventral plan viewrespectively of one embodiment of a device according to the invention;

FIGS. 2a and 2b illustrate a cross-sectional and ventral plan viewrespectively of an alternate embodiment of such a device;

FIGS. 3a, 3b and 3c illustrate yet another embodiment of a pinpointmicro-container device with introducer;

FIGS. 4a, 4b and 4c illustrate still another embodiment of the inventionwith a refillable micro-container and detachable fixation means; and

FIG. 5 is a graph showing the collagenase diffusion rate across thesclera.

DETAILED DESCRIPTION OF THE INVENTION

The device of this invention will now be illustrated with reference tothe drawings, which are not limiting but merely illustrative of theinvention.

Referring now to FIGS. 1a and 1b, there is shown a device 1 according tothe invention in the shape of a disc 2 having a diameter of about 5 mm.The height of the disc (a) is approximately 1 mm. From the ventral side4 of the disc there extends a cylinder 3 having an outer diameter (x) ofapproximately 1 mm. The cylinder 3 extends about 0.5 mm (b) beyond theventral side 4 of the disc. A bore 5 in the cylinder 3 has a diameter(y) of about 0.7 mm, which extends about 0.5 mm (c) into the disc 2 toprovide a micro-container 6 with an internal volume of about 0.38 mm³.The bottom edge 7 of the cylinder wall 3 is bevelled, coming to a pointat its aperture in order to provide minimum contact surface where themicro-container contacts the eye.

In general, the micro-container should have a volume of preferably about0.2 mm³ to 1.5 mm³ with a bore depth of about 0.5 mm to 1.5 mm. Theaperture wall thickness should provide little surface contact with thesclera, and should preferably range from about 0.01 mm to 0.25 mm.

The device is used as follows. A suitable pharmaceutical composition, asfor example collagenase either as a lyophilised powder or an aqueoussolution containing a calcium salt, is introduced into the cylinder bore5 which serves as a micro-container 6. The ventral side 4 of disc 2 ischarged with histoacryl medical glue, which will adhere the disc to thesclera of the eye. The ventral surface of the disc is preferably maderough, so that the glue can better adhere thereto. With forceps or othergripping means, the disc is manipulated under the conjunctiva and overthe surface of the scleral limbus of the eye, so that themicro-container 6 is brought into pinpoint contact with the desired areaon the sclera and the glue contacts the sclera only adjacent themicro-container 6, assuring the adherence of the device 1 to the sclerafor a given amount of time. The micro-container is thus in directcontact with the sclera in place. A collagenase composition can beapplied via the micro-container at the sclero-corneal junction toaugment sclero-limbal permeability and lower intra-ocular pressure.

Turning now to FIGS. 2a and 2b, there is shown another embodiment of thedevice 11 comprising a disc 12 and cylinder 13 which provides amicro-container 16. The bottom edge of the bevelled wall 17 provides asharp aperture corresponding approximately to the outer diameter of thecylinder. In this embodiment, the ventral side 14 of the disc 11comprises a recess 18 for holding the glue. It should be noted that thebevel 17 is at such an angle so that the aperture 15 has a largerdiameter than the micro-container 16. This embodiment enables fixing thedevice on the sclera with minimal chances of the glue oozing into themicro-container 16 under the cylinder walls, since the glue can flowinto the recess 18 and because of the sharp barrier formed by thebevelled edge.

In FIGS. 3a, 3b and 3c there is shown another embodiment wherein thedevice comprising a disc 31 having an integral micro-container 36 withan introducer handle 38 releasably attached to the disc 31 with which tomanipulate the micro-container into position on the sclera. Medical glue20 is spread on the ventral side 34 of the disc. This device is used asfollows. A 1 cm slit is made in the conjuctiva 1 cm posterior to thelimbus and the micro-container 36 is inserted therein with theintroducer 38. The micro-container 36 is placed in position at thelimbus (when treating glaucoma) and the introducer handle 22 is removedwhile pressing down at the depression 39 of the dorsalodise of the disc31 with a finger and pulling the introducer 38 back, thereby removingshield 21 from under the micro-container 36 and glue 20, thus contactingthe sclera directly with the glue.

FIGS. 4a, 4b and 4c show yet another embodiment of the invention whichcomprises a micro-container consisting of a cylinder 41 having an outerdiameter of about 2.0 mm and a threaded bore 42 of about 1.2 mm. Agroove 43 circumferentially encompasses the cylinder 41. Extending fromthe cylinder 41 there is a narrower cylinder 44 having an outer diameterof about 1.0 mm and internal diameter 0.7 mm, providing amicro-container 45. The inner space of cylinder 44 is continuous withthe threaded chamber 42 and provides a micro-container 45. A screw cap48 (shown in FIGS. 4b and 4c) having a vertical groove 47 extendingpartly along its outer edge, is threaded through bore 42, thus fixingthe volume of the micro-container 45. A depression 49 serves to insert ascrewdriver for turning the screw cap 48. A silicone ring (not shown) isplaced around the cylinder 41 and held in place in groove 43. Thissilicone ring serves as the fixation element to whose ventral side gluecan be applied for adhering the micro-container to the sclera. Theadvantage of this particular embodiment is that the pharmaceuticalcomposition can be introduced into the micro-container 45 through theopen threaded bore 42 even after the device is glued in position, andsubsequently capping the bore 42 with screw 48 of FIG. 4b. The purposeof groove 47 is to provide a vent for letting air out of the bore 42while the screw 48 is being tightened to seal bore 42 completely withthe screw 48. This embodiment also enables changing medication, forexample collagenase neutralizing agent, or dosage while the device isstill adhering to the sclera without removing it.

It is of course understood that although the examples use medical gluefor adhering the fixation elements to the sclera, it is possible to usealternative methods, such as vacuum or thirsty glass for fixation means.In the latter case these would not be recessed with respect to themicro-container aperture, but would be flush therewith to be placeddirectly on the sclera.

Any pharmaceutical composition for treating the eye can be used with thedevice of the present invention. Such compositions may be liquids, gelsor solids such as lyophilised powders. We have found this deviceparticularly useful for the treatment of glaucoma by pinpointapplication of collagenase.

The method of the instant invention is exemplified by experimentsconducted on rabbits. The animal is stabilized in an examinationrestrainer cage, a pediatric lid retractor is introduced for eyelidstabilization and one drop of 0.4% oxybuprocain is instilled topically,for providing appropriate anesthesia to inhibit the brisk motion due tothe pain reflex during application. For augmentation of aqueous outflowwith consequent drop of intraocular pressure, a peritomy of about 3 mmis efectuated at the limbus and the said device, loaded with the saidcollagenase, is gently introduced on the limbus or within 1 mm from itto adhere by the said methods, and contact the limbal collagen for therequired time interval in order to attain the necessary tissue thinning.Applications of the loaded device or direct injection of the saidcollagenase more than 2 mm posteriorly from the limbus, shows the sametissue alteration but without any influence on the intraocular pressure.Due to the safety and the ease of the invented method, reapplication canbe carried out after immediate or delayed time intervals from the firstintervention, if the desired effect on intraocular pressure is notachieved or diminishes too soon, respectively. Delivery and redeliveryof the said collagenase to contact the tissue while the device is in astate of adherence is possible by filling-up the micro-container whenusing one of the invented embodiments.

The depth of enzymatic tissue degradation can be monitored throughoutthe period of application by means of ultrasonography, permittingremoval of the device at the proper time. However, it is disclosed inthe present invention that contact of 200 μg collagenase for 4 h' is theoptimal time for achievement of the proper limbal thinning which allowsaugmented permeability and increment of outflow with consequent drop ofintraocular pressure.

The tissue alteration caused by the application of the said collagenaseby the method of the invention is demonstrated by histology of thinsections of the site of application at different time intervals afterthe application and found to have a unidirectional transscleralvectorial propagation of the collagenase effect, creating a base-up coneshape smooth walls tissue alteration. The duration of this effect ismonitored by intraocular pressure measurements at due time intervals.

In the preferred embodiment of this invention, collagenase produced bythe bacterium Clostridium histolyticum (clostridiopeptidase A) is used.This type of collagenase is commercially available from AdvancedBiofactures Corporation and is sold as Collagenase (form III) and fromWorthington, where it is sold as Collagenase (CLSPA). However anycollagenase composition chromatographically purified by anion exchange(DE-52) and further by molecular sieving, having the main sixcollagenase isoenzymes, exhibiting its main activity on insolublecollagen substrate and having negligible activity of other proteasesespecially caseinase, are suitable and may be utilized in the practiceof this invention. The said pharmaceutical composition can be used withthe said device of the present invention. Such compositions may be abuffered solution of calcium acetate 10-33 mmolar, pharmacological gelswell known in the art or, as preferred in the instant invention,lyophilized powders.

The cornea and the trabecular meshwork are lined with endothelial cellsnot allowing compounds with molecular weight greater than 6200 kDa topass through the cell wall and contact the collagen fibers underneath.When the said collagenase, which has a molecular weight much greaterthan 6200 kDa, penetrates the anterior chamber in the amounts suggestedfor the said treatments, it does not significantly affect thesestructures. Another exposed structure in cases of penetration into theanterior chamber is the collagen rich iris protected evenly anteriorlyby the pigmented anterior border layer. The risk of passage of eventualpenetrated collagenase solution from the anterior chamber to thecollagen-rich structures in the posterior segment is minimal since thethermal circulation of the aqueous washes the collagenase solution outof the eye through the new path formed by the treatment itself.Moreover, the aqueous was found to have intrinsic inhibitory propertieson the collagenase used.

It is preferred that the flush solution used after removal of themicro-container to remove any trace of collagenase activity have aphysiologic compatible pH of 7.40 and contain calcium chelator EDTA 0.5%as routinely used in ophthalmology. Rinsing should be carried out withan abundant amount of the said inhibitory solution, immediately afterdevice removal, on all subpalpebral regions with special attention tothe site of application. Moreover, the aqueous penetrating now in anenhanced manner through the site of application is capable of removingresidual detrimental collagenase from the site and is noted to haveintrinsic inhibitory properties upon the said collagenase activity.

The following experiments conducted in vitro on bovine sclera or in vivoon rabbits are exemplary. In all experiments the pharmaceuticalcomposition shall refer to collagenase (CLSPA manufactured byWorthington). When used as solution the pharmaceutical composition wasdissolved in buffer containing 50 mM Tris HCl, 10 mM Ca acetate pH 7.4.The device comprises the micro-container loaded with the saidcollagenase as solution, as lyophilized powder and as chemicallyimmobilized enzyme, however it is within the scope of this invention toinclude other medications and pharmaceutical preparations used assolutions, lyophilized powder and chemically immobilized activesubstances. It is also within the scope of this invention to includeother devices providing limited restricted and isolated area of contactbetween chemical composition and tissue.

EXPERIMENT 1

The micro-container of the device in FIG. 1 was loaded with variousconcentrations of collagenase solution (up to 50 μg/0.5 μl--see FIG. 5),the ventral face of the metal plate around the micro-container wassmeared with medical hystoacryl glue and applied to buttons of bovinesclera to adhere and contact the scleral collagen. After incubation inhumid conditions at 37° C. for 16 hours, the device was removed and thetissue was rinsed with a solution of 70 mM EDTA. The scleral button wasmounted in a "tissue permeability chamber" under pressure of 20 mmHg.The flow of liquid (phosphate buffered saline--PBS) through the scleralbutton was monitored by recording the waterhead decrease (initially 20mmHg) in a calibrated pipet above the sclera, as well as measuring thevolume collected underneath in the collector and was found to be1.2-11.5 μl/h in accord with amounts of collagenase applied (0.5 μl,6.25-50 mg/ml). The transscleral flow was calculated as 0.7 μl/μgcollagenase/hour (FIG. 5). This result shows a substantial increase ofthe transscleral flow reached within the range of applicable amounts ofcommercial collagenase in comparison to the basic flow of 1 μl/hourthrough a control, enzymatically untreated, scleral button. The sites ofthe pinpoint application of collagenase were subsequently examined andthese were found to have bore marks of 1 mm in diameter anddeepness >75% of the tissue thickness. The bore had cone shape geometrywith a round base at the site of application and smooth walls thatgradually narrowed in the direction of the propagation of thecollagenolytic effect. There was no lateral spreading on the surface and"intra-tissue" of the action of collagenase. This may be due to thescleral architecture characterized by highly organized collagenstructure, by the design of the device and by the mode of isolation ofapplication with the hystoacryl glue, which causes the unidirectionaltransscleral vectorial propagation of the enzymatic degradation.

EXPERIMENT 2

The micro-container of FIG. 1 was applied to the limbus of the right eyeof an eight month white New Zealand rabbit. After 20 hours the animalwas repositioned in the examination case for inspection and ocularmanipulation. Temporary chemosis (conjunctival inflammatory response)was mild and diffuse and gradually disappeared within one week. Therewas no corneal damage, the anterior chamber was quiet, the pupilaryresponse was normal and there was no change in the pre-applicationintraocular pressure. The device was removed with a Macpherson forceps,no scleral alteration was observed and the conjunctiva was repositedwithout sutures. This experiment shows the safety of using the device ofthis invention.

EXPERIMENT 3

To the limbus of each left eye of 3, four and half kg white New Zealandrabbits, 180±50 μg of the pharmaceutical composition of Experiment 1 wasapplied by means of the method of the invention through the said device(FIG. 2). As control, a similar device was mounted to the right eye bythe said method without the pharmaceutical composition. Thepre-application biomicroscopic examination was normal and theintraocular pressure was 15 mmHg. After 4 hours the device was removedand a survey of the eyes revealed similar features in all animals: Inthe left eye a mild conjunctival inflammatory response, and a brownishbore 1 mm in diameter. In the right eye a similar conjunctivalinflammatory response was noticed without any scleral alteration(Example 2). The site of application was flushed with the saidinhibitory solution. On follow-up the intraocular pressure in the lefteye decreased by more than 50% in comparison to the right eye, duringthe first week. A meaningful decrease of intraocular pressure of about30% continued for more than one month.

EXPERIMENT 4

The limbus of two rabbits was injected in about half the width of thetissue, with a solution (1.0 mg/ml) of the pharmaceutical composition ofExperiment 1 (0.5 μl) by means of a Hamilton syringe (No. 7000,5). Inthe other eye buffer was injected in the same manner. On examinationduring the first day after the injection, both eyes were quiet and onlyin the eye injected with collagenase an area of filtration was formed bylysis of the collagen of a diameter of more than 0.5 cm invading thecornea where it appears cloudy and opaque. The effect of pressuredecrease, of about 30%, was limited to one week. This direct limbalinjection of collagenase was carried out in order to compare it with themethod of the invention showing that even when small amounts wereinjected to the eye coat, the extent of spreading of collagen lysis isnot controllable and the tissue was affected far beyond the site ofinjection extending to the cornea as well.

EXPERIMENT 5

In order to test the distance of effective application posterior to thelimbus, the said device loaded with the same pharmaceutical compositionas in Experiment 1 (1 μl, 50 mg/ml) was positioned to contact thescleral collagen with the orifice of the micro-container 3 mm posteriorto the limbus. After 20 hours the device was removed disclosing an areaof similar changes as previously described (Experiment 3) but withoutnoting any difference in intraocular pressure from the untreated eye.Hence, increased permeability at a site distant from the limbus does notpermit outflow of aqueous and is not useful for treatment of glaucoma.

EXPERIMENT 6

Direct injection of the pharmaceutical composition (0.05 μl 40 mg/ml)into the anterior chamber was carried out by means of a Hamilton syringe(No. 7000,5). The eye was inspected by biomicroscopy and the followingminor changes were noted: a thin stream of blood, probably from theaqueous veins that in the rabbit are filled with blood, was observed onthe corneal endothelial layer immediately after the injection. On thefirst day of follow-up, moderate inflammatory reaction was observed;ciliary injection restricted to the area of application, engorgement ofiris vessels, with flare and cells in the normally deep anteriorchamber. These signs decreased gradually and finally disappeared afterone week. It is concluded that penetration into the anterior chamberwith introduction of small amounts of the said pharmaceuticalcomposition, did not elicit a major pathologic response.

EXPERIMENT 7

In order to test the possibility of delivery of collagenase solutionwhile the device is in a state of adherence, a device (FIG. 4) wasapplied to the limbus of the left eye of a rabbit containing 50 μg oflyophilized collagenase powder. After two hours the stopper (4b) wasremoved and an additional amount of collagenase (0.5 μl 200 μg/μl) wasinjected by means of a Hamilton syringe into the micro-container, thenthe stopper was repositioned. The loaded device was left to contact thesclera for another two hours and was then removed. The scleralalterations observed were similar to those observed after a singleapplication of the device with a sufficient amount of collagenase. Thedrop of intraocular pressure was similar to that seen after a singleapplication. This data indicates that application and reapplicationwhile the device is in a state of adherence is feasible.

EXPERIMENT 8

The possibility of a repeated intervention using the method of theinvention was examined. Reapplication of the device was carried out tothe limbus of the left eye of one rabbit in a location adjacent the areatreated several months previously by the same method after return of theintraocular pressure to values of the control eye. The micro-containerwas loaded with 165 μg of the said. pharmaceutical composition and theduration of application was 4 hours. The result was similar to the firstapplication both macroscopically and functionally. The pressuredecreased to 50% in comparison to the right eye for one week, thenremained decreased to 30% for more than one month.

While the above experiments were conducted on rabbits, the results areindicative of what can be anticipated with all mammals. This inventionmay be practiced on any animal including a human.

A rabbit's occular region is similar to a human's in that both containmany collagen rich structures. The rabbit cornea, lens and anteriorchamber is similar. The sclera's collagenous composition, although beingsimilar, is thinner both in the single collagen fiber and in the totaltissue thickness. Accordingly a different optimalization of amounts andtime of application may be required in humans using the same device bythe same method of enzyme-tissue contact, limitation and focalization.

In view of the preceding description, further modifications andalternative embodiments of the instant invention will be apparent tothose skilled in the art. Accordingly, the preceding description is tobe construed as explanatory and illustrative only and is for the purposeof teaching and enabling those skilled in the art to practice thisinvention. It should be understood that the suitable amount ofpharmacological composition and the period of application may vary, aswell as the predetermined area of application.

We claim:
 1. A device for pinpoint application of micro-quantities of apharmaceutical composition to a preselected portion of the outer hardcoat of the eye,the device comprising a micro-container with fixationmeans for reversible attachment of said micro-container to the outerhard coat of the eye adjacent to the preselected portion thereof, saidmicro-container comprising a pharmaceutical composition delivery borewhose aperture is adapted to contact the preselected portion of theouter hard coat of the eye so as to expose only the preselected portionthereof to the pharmaceutical composition.
 2. A device according toclaim 1, wherein the micro-container and fixation means comprise asingle unit.
 3. A device according to claim 1, wherein the fixationmeans is separable from the micro-container.
 4. A device according toclaim 1, wherein the micro-container comprises a cylinder with a boreabout 0.5 to 1.5 mm.
 5. A device according to claim 1, wherein themicro-container has a volume of about 0.2 mm³ to about 1.5 mm³.
 6. Adevice according to claim 1, wherein the micro-container comprises aremovable stopper which enables filling the micro-container while it isin the state of adherence to the eye surface.
 7. A device according toclaim 1, wherein the thickness of a micro-container wall at its apertureis between 0.1 mm to 0.25 mm.
 8. A non-invasive method for applyingpharmaceutical compositions to a preselected portion of the outer hardcoat of an eye at pinpoint locations, comprising the followingsteps:providing a micro-container containing an amount of apharmaceutical composition and having a pharmaceutical compositiondelivery bore; and reversibly attaching the micro-container to the outerhard coat of the eye so as to locate the delivery bore in contact withsaid preselected portion and thereby deliver the pharmaceuticalcomposition solely to said preselected portion.
 9. A method according toclaim 8, wherein the micro-container is first placed on the desiredlocation of the eye surface and fixed thereto and subsequently apharmaceutical composition is introduced into the micro-container.
 10. Amethod for treating glaucoma, comprising administering, at pinpointlocation on the limbus of the eye for a given time, a sufficient amountof collagenase composition to effect limited controlled tissuedegradation, thereafter neutralizing the collagenase and rinsing theeye, thereby decreasing the intraocular pressure.